Unit: Immuno-Parasite Signalling

Director: Gordon Langsley


Left, Hoechst DNA stain of infected TpMB2 cell nucleus (large blue) and parasite multinucleated macroshizont (small blue dots, indicated with arrow). JAK2 inhibition leads to both Annexin V-positivity of B cell plasma membrane (middle) and nuclear fragmentation (right).

Theileria-induced transformation of bovine leukocytes is unique in that it is entirely reversible as upon drug-induced parasite death the leukocyte reverts to a quiescent cell that will die from apoptosis unless further stimulated. The reversible nature of the transformed state provides a powerful model to study host-parasite relationships in the context of leukocyte activation and we use this model to address three basic questions: 1) Leukocyte survival i.e. how does the parasite prevent the activated leukocyte from dying of apoptosis? 2) Leukocyte proliferation i.e. how does the parasite induce the uncontrolled proliferation of its host cell? 3)

We have approached the question of obligatory parasitism via the study of host-parasite communication, and this in practical terms we have defined as the study of host-parasite signalling interactions. As Plasmodia and Theileria communicate with their host cells by secreting parasite proteins into the host cytosol, and in the case of P. falciparum even to the host cell surface, we have also characterised the small GTPases (Rabs) that regulate the crucial steps of vesicular traffic between the parasite and its host.

For the last few years our mostly biochemical and cell-biological approaches have focussed on the signal transduction pathways that are activated by Theileria infection of lymphocytes and we have determined the role these pathways play in inducing lymphocyte survival and metastasis. A good example being our recent paper (Baumgartner at al, 2003 Blood 101: 1874-81) describing the permanent activation of Hck (a Src kinase) by constitutive exclusion of its negative regulator Csk from the Rafts of Theileria-transformed B cells. Hck activation contributes to induction of the AP-1 transcription factor and we have demonstrated previously that this is involved in metastasis of Theileria-infected leukocytes.

Concomitantly, we have continued our study of both parasite and host Rab GTPases and for a description of the P. falciparum Rabs family one should refer to our recent largely bioinformatics based paper (Quevillion et al, 2003 Gene 306: 13-25). In Theileria-infected lymphocytes we have shown that one consequence of AP-1 activation is enhanced transcription of the rab11 gene (Rab11 is specific for recycling endosomes) that leads to accelerated turnover of the transferrin receptor (TfR) that is associated with Theileria-induced uncontrolled lymphocyte proliferation.

Having focussed on the signals leading to AP-1 induction we have now turned to isolating the Theileria gene(s) responsible for its activation, as these represent parasite virulence gene(s). We have developed genetic strategies based on complementation and to this end have constructed a Theileria-specific cDNA library. In collaboration with Genopôle-Pasteur we have sequence 10,000 random cDNAs all of which were of parasite origin. This EST project forms part of a larger Sanger Centre led genome project, where our contribution was transcription profiling of the transforming macroschizont stage. The genomes of Theileria annulata (Sanger Centre) and T. parva (TIGR) have been completely sequenced and annotated (the Genome Centres have decided to submit back-to-back papers before the end of the year) and this allows us to use comparative bioinformatics to identify parasite virulence factors. As an example, we can subtract >from the predicted 3, 800 Theileria ORFs the 1, 600 orthogues common to Plasmodia. From the remaining 2, 200 candidates we can further subtract all those (brake in synteny) genes that vary between T. annulata and T. parva, as these are fast-evolving and under immune pressure i.e. they represent good vaccine, but poor oncogene candidates. Next, we can positively select all those that scored with our transforming macroschizont ESTs and negatively those that were positive with piroplast ESTs, as piroplasts infect erythrocytes. Finally, we can positively select all genes encoding a signal peptide, as we have presumed (see above) that virulence factors are secreted. This comparative bioinformatics selection leaves us with just 244 candidates that can be tested in pools or individually for their ability to induce AP-1 activation.

In summary, we exploit the comparative biology of host-parasite interactions via the cell biology of signalling in Theileria- and Plasmodia-infected cells. Due to our involvement in the Sanger Centre/Wellcome Trust genome project and our construction of the Theileria-specific cDNA library, we are currently using complementary genetic approaches to isolate the virulence genes responsible for activation of the signalling pathways that we have previously characterised.

Photo :

Left, Hoechst DNA stain of infected TpMB2 cell nucleus (large blue) and parasite multinucleated macroshizont (small blue dots, indicated with arrow). JAK2 inhibition leads to both Annexin V-positivity of B cell plasma membrane (middle) and nuclear fragmentation (right).

Keywords: parasite, Theileria, signalling, transformation, apoptosis, metastasis

Activity Reports 2003 - Institut Pasteur

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